Prostate cancer is the most common non-dermatological malignancy in the United States, the second leading cause of cancer-related death in American males, and has one of the strongest relationships with age of any human malignancy. One of the most common therapies for advanced prostate cancer is androgen deprivation via chemical or physical castration. Unfortunately, most patients relapse and continue to experience debilitating bone pain, urinary symptoms, and sexual dysfunction. At this stage, the cancer is termed androgen-independent or castration resistant (CRPC);however, it has been shown that this stage of cancer remains dependent on androgen receptor (AR) signaling. One of the proteins implicated in this enhanced AR transcriptional activity is Vav3, a Rho GTPase guanine nucleotide exchange factor. Vav 3 is up-regulated both in vitro and in vivo in androgen-independent human prostate cancer and in the Nkx3.1;Pten mutant mouse model of age-related prostate cancer. Vav3 enhances AR activity at both physiological as well as at subnanomolar androgen concentrations, similar to prostatic microenvironment levels demonstrated in CRPC. Thus, elevated levels of Vav3 seem to be pivotal in the continued AR signaling in androgen-independent cancers. In the presence of androgen, Vav3 potentiation of AR transcriptional activity requires the Vav3 pleckstrin homology (PH) domain, but not guanine nucleotide exchange factor (GEF) activity. Furthermore, preliminary data suggests that a mutation in the PH domain causes Vav3 to be excluded from nuclei and incapable of recruitment to the androgen responsive element in an AR target gene. On the other hand, constitutively active Vav3 has been shown to enhance ligand-independent AR activation via GEF activity and the Rho GTPase Rac1. Through an approach utilizing separation-of-function Vav3 mutants, contributions of ligand-independent Vav3 GEF activity and ligand-dependent Vav3 coactivation of AR can be determined. In this study, various Vav3 mutants will be stably transfected into the Vav3 deficient prostate cancer cell line LNCaP and their gene expression profiles will be examined in order to identify the molecular pathways through which Vav3 enhances AR transcriptional activity. The Vav3 separation-of-function mutants will then be orthotopically injected into the prostates of immunocompromised mice in order to better recreate the prostatic microenvironment. These cells will be evaluated for differences of in vitro and in vivo invasion and cell growth in order to examine the role of distinct AR activation pathways in age-related prostate tumor formation and metastasis. In addition, Nkx3.1;Pten mutant mice, which are faithful models of aging-dependent prostate cancer, will be examined following mating with available Vav3 null mice to define the contribution of Vav3 signaling in this model. Identified sentinel gene expression pathways that are up or down-regulated in various forms of Vav3 stably transfected prostate cancer cells will be compared to the resulting expression patterns in both the Nkx3.1;Pten and Nkx;Pten;Vav3 knock out mouse models. Furthermore, by comparing the sentinel gene clusters identified via genetic profiling to the prognostic outcomes of the age-dependent tumors in the mutant mice, novel Vav3 signaling pathways may be identified and thus novel diagnostic markers uncovered. PUBLIC HEALTH RELEVANCE: Prostate cancer is the most common non-dermatological malignancy in the United States, the second leading cause of cancer-related death in American males, and has one of the strongest relationships with age of any human malignancy. Androgen receptor (AR) and its co-activating proteins play a key role in increasing prostate cancer's malignancy, metastatic potential and debilitating physical symptoms. This research endeavors to delineate the pathways behind the ability of an AR activating protein (Vav3) to increase the malignancy of prostate cancer with the eventual aim of providing a new target for a prostate cancer drug and a prostate cancer diagnostic marker.